Diabetes often begins with insulin resistance brought on by weight gain. Beta cells of the islets of Langerhans compensate for insulin resistance by increasing their mass and insulin secretion. In diabetics, compensation fails so that insulin secretion is inadequate. Diabetics also experience improperly elevated glucagon secretion from islet alpha cells. As glucagon stimulates release of glucose from liver stores, excessive glucagon exacerbates hyperglycemia caused by insufficient insulin. This effect is now recognized as important in development of the disease. Endocrine cell dysfunction may be caused by elevated glucose (glucotoxicity). Greater understanding of glucagon and insulin secretion in normal and diabetic conditions is of fundamental importance in determining root causes of diabetes and developing treatments. Understanding beta cell proliferation is significant because gaining control of this process would enable prevention or treatment of diabetes. Studies of secretion and proliferation are critically dependent upon instrumentation that can detect relevant biochemical dynamics. Our objective is to develop new analytical methods suitable for measuring chemical events related to peptide secretion from islet cells. Our first aim is to develop a microelectrode that can detect glucagon secretion at the level of single exocytotic events from single cells. Our second aim is to develop metabolomic methods to measure metabolites in beta and alpha cells. We expect this method to provide significant insights as both cell types use glucose metabolism to drive secretion. Impaired metabolism is likely behind defects in secretion. As purified cells can only be obtained in small quantities, to achieve such measurements we must develop miniaturized tools to culture and process the cells for analysis. We will use a novel microfluidic system to control cell environment and perform the necessary extraction steps on 5,000 cells. The resulting extracts will be analyzed by miniaturized chromatography columns coupled to mass spectrometry. Our third aim is to develop a microscale protein assay system for use in islet cell proliferation studies. The standard tool for dissecting protein signaling involved in proliferation is western blotting; however, this method is slow and ill-suited to measuring the many different proteins involved simultaneously. To improve research in this area, we will develop a microfluidic western blot that automates and speeds up the process while enabling multiplexed protein assay on single islets. Our final aim is to deploy these new methods in fundamental studies of endocrine cell secretion and proliferation. We will characterize hormone secretion at the single cell level and then determine how glucotoxicity affects this process. In parallel, we will assess how the alpha and beta cell metabolome responds to glucose and how this response is altered by glucotoxicity. The combined studies will allow us to unravel mechanisms of secretion and how it is affected in diabetic conditions. We will also determine protein pathways activated for proliferation and how they are altered in glucotoxic environments.